Methods for the selection of aptamers

ABSTRACT

The present invention generally relates to methods for selecting aptamers. More specifically, the invention provides methods for the selection of at least one aptamer for use in combination with another epitope binding agent such as another aptamer, an antibody, or a double stranded nucleic acid. The invention also encompasses methods for simultaneously selecting at least two aptamers that each recognize distinct epitopes on a target molecule.

FIELD OF THE INVENTION

The present invention relates to methods for selecting aptamers. More specifically, the invention provides methods for the selection of aptamers for use in combination with another epitope binding agent such as another aptamer, an antibody, or a double stranded nucleic acid. The invention also encompasses methods for simultaneously selecting at least two aptamers that each recognize distinct epitopes on a target molecule.

BACKGROUND OF THE INVENTION

The ability to detect, label, and track a molecular target is a powerful research tool. To date, antibodies are the typical reagents used for such research. Antibodies have high specificity for their target, and often can be used for in vivo as well as in vitro applications. Producing antibodies, however, is a sensitive, expensive, and time-consuming endeavor. Polyclonal antibodies, although highly specific, are limited in supply. Monoclonal antibodies, while extremely useful for molecular biology techniques, require labor intensive, diligent cell culture work. Furthermore, if a specific antibody will be used for in vivo human studies, the antibody may require modification to avoid triggering an unwanted immune response. Despite these drawbacks, antibodies have remained the state of the art in many biological research fields.

Aptamers, or target specific nucleic acid sequences, offer an alternative to antibodies. The advent of in vitro selection of aptamers was key to demonstrating their utility. Aptamers can be selected to recognize virtually any target, and they can be synthetically engineered, greatly reducing cost and time when compared to producing antibodies. The most common method for selecting aptamers is referred to as Selex (Systematic Evolution of Ligands by Experimental Enrichment). In general, the Selex process selects target specific aptamers from a pool of randomly generated nucleic acid oligonucleotides through several rounds of selection and amplification. Despite the fact that aptamers are less expensive to make, do not require animal facilities to produce, and can be selected with simple molecular biology techniques, they remain in the background of biological research. A need exists for better aptamer selection methods, which will further increase the advantage of aptamers relative to antibodies. Moreover, although the Selex method has advanced the field of aptamer selection with respect to the selection of one aptamer, a need exists for methods involving the simultaneous selection of two or more aptamers that recognize distinct epitopes on a target molecule.

SUMMARY OF THE INVENTION

Among the several aspects of the invention, therefore, is a pair of nucleic acid constructs comprising a first nucleic acid construct and a second nucleic acid construct. The first nucleic acid construct comprises: A-B-C-D;

-   -   the second nucleic acid construct comprises:         E-F-G-H;     -   wherein:         -   A, C, E, and G are each different DNA sequences from about             10 to about 30 nucleotides in length, A and C together             comprising a sequence to prime a polymerase chain reaction             for amplifying a first aptamer sequence, and E and G             together comprising a sequence to prime a polymerase chain             reaction for amplifying a second aptamer sequence;         -   B is a single-stranded random nucleotide sequence from about             20 to about 110 nucleotides in length that contains specific             sequences binding to a first epitope of the target molecule;         -   D and H are a pair of complementary nucleotide sequences             from about 2 to about 20 nucleotides in length, wherein D             and H have a free energy for association from about 5.5             kcal/mole to about 8.0 kcal/mole at a temperature from             approximately 21° C. to about 40° C. and at a salt             concentration of approximately 1 mM to about 100 mM; and         -   F is a single-stranded random nucleotide sequence from about             20 to about 110 nucleotides in length that contains specific             sequences binding to the second epitope of the target             molecule.

Another aspect of the invention provides a method to select at least one aptamer in the presence of an epitope binding agent construct. Alternatively, the invention encompasses a method for simultaneously selecting at least two aptamers.

Other aspects and features of the invention will be in part apparent and in part pointed out hereinafter.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts a series of schematics illustrating molecular biosensors for detecting target molecules. (A) Variant of the design for target molecules lacking natural DNA binding activity. The biosensor in this case will be composed of two aptamers developed to recognize two different epitopes of the target molecule. (B) Variant of the design for a target molecule exhibiting natural DNA binding activity. The biosensor in this case will be composed of a short double-stranded DNA fragment containing the DNA sequence corresponding to the DNA-binding site and an aptamer developed to recognize a different epitope of the target molecule.

FIG. 2 depicts a series of schematics illustrating methods for preparing aptamers according to the invention. (A) Selection of an aptamer in the presence of a known aptamer construct. The process is initiated with a nucleic acid construct, an aptamer construct (composed of an known aptamer (thick black line), a linker, and a short oligonucleotide sequence (hatched bar)), and the target molecule (grey). The hatched bars depict complementary short oligonucleotide sequences. (B) Simultaneous selection of two aptainers that bind distinct epitopes of the same target molecule (grey). The process is initiated with two types of nucleic acid constructs (the primer1-2 construct and the primer3-4 construct) and the target molecule. The hatched bars depict short complementary sequences at the end of the two types of nucleic acid constructs. (C) Alternative design for simultaneous selection of two aptamers that bind distinct epitopes of the same target molecule (grey). An additional pair of short oligonucleotides (hatched bars) connected by a flexible linker is present during the selection process. These oligonucleotides will be complementary to short oligonucleotide sequences at the end of the nucleic acid constructs (in primer 1 and primer 4). Their presence during selection will provide a bias towards selecting pairs of aptamers capable of simultaneously binding to the target molecule. Before cloning of the selected nucleic acid constructs the pairs of selected sequences will be ligated to preserve the information regarding the preferred pairs between various selected constructs. (D) Selection of an aptamer in the presence of a known antibody construct. The process is initiated with a nucleic acid construct, an antibody construct (composed of a known antibody (thick black line), a linker, and a short oligonucleotide sequence (hatched)), and the target molecule (grey). The hatched bars depict complementary short oligonucleotide sequences. (E) Selection of an aptamer in the presence of a known double-stranded DNA construct. The process is initiated with a nucleic acid construct, a double-stranded DNA construct (composed of an known double-stranded DNA sequence, a linker, and a short oligonucleotide sequence (hatched)), and the target molecule (grey). The hatched bars depict complementary short oligonucleotide sequences.

FIG. 3 is a schematic illustrating various formations of molecular biosensors that can be made utilizing aptamers selected according to the invention.

FIG. 4 is a schematic illustrating the method of the invention.

FIG. 5 summarizes the selection of an aptamer that binds to thrombin at an epitope distinct from the binding site of the G15D aptamer. (A) An illustration of the reagents used to begin the process of selection. (B) The graph indicates the increase in thrombin binding with successive rounds of selection. (C) The sequences represent aptamers developed after 12 rounds of selection.

FIG. 6 The demonstration of the functional thrombin sensor comprising Texas Red-labeled THR27 and fluorescein-labeled THR35 or THR36 (both contain the sequence corresponding to that of clones 20, 21, 24, and 26 of FIG. 5C). The fluorescence image represents the specificity of either 20 nM (panel A) or 100 nM (panel B) of the indicated biosensor.

FIG. 7 summarizes the simultaneous selection of two aptainers that bind to thrombin at distinct epitopes. (A) An illustration of the reagents used to begin the process of selection. (B) The graph indicates the increase in thrombin binding with successive rounds of selection. (C) The sequences represent aptamers developed after 13 rounds of selection.

FIG. 8 summarizes the selection of an aptamer that binds to CRP at an epitope distinct from the DNA-binding site. (A) An illustration of the reagents used to begin the process of selection. (B) The graph indicates the increase in thrombin binding with successive rounds of selection. (C) The sequences represent aptamers developed after 11 rounds of selection.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Methods for efficiently selecting aptamers have been discovered. The invention is directed to methods for simultaneously selecting two or more aptamers that each recognize distinct epitopes on a target molecule. Utilizing the method of simultaneous aptamer selection typically cuts the time of selection in half compared to methods involving sequential selection of two aptamers using Selex. Alternatively, the invention is directed to selecting at least one aptamer in the presence of an epitope binding agent construct. The aptamer and epitope binding agent construct also each recognize distinct epitopes on a target molecule. In each method of the invention, novel nucleic acid constructs are utilized to facilitate selection of aptamers having the desired epitope binding characteristics. In each aspect of the invention, the nucleic constructs comprise the aptamer or aptamers selected by the method of the invention. Advantageously, the methods of the invention may be utilized to select aptamers to construct various molecular biosensors as illustrated in FIG. 3.

(A) Method for Selection of an Aptamer in the Presence of an Epitope Binding Agent Construct

One aspect of the invention encompasses a method for selecting an aptamer in the presence of an epitope binding agent construct. The aptamer and epitope binding agent construct are selected so that they each bind to the same target at two distinct epitopes. Typically, the method comprises contacting a plurality of nucleic acid constructs and epitope binding agent constructs with a target molecule to form a mixture. The mixture will generally comprise complexes having target molecule bound with nucleic acid constructs and epitope binding agent constructs. According to the method, the complex is isolated from the mixture and the nucleic acid construct is purified from the complex. The aptamer selected by the method of the invention will comprise the purified nucleic acid construct.

In this method of selection, a plurality of nucleic acid constructs is utilized in the presence of the epitope binding agent construct to facilitate aptamer selection. The nucleic acid constructs comprise: A-B-C-D

The epitope binding agent construct comprises: P-Q-R

-   -   wherein:         -   A and C are each different DNA sequences from about 10 to             about 30 nucleotides in length, A and C together comprising             a sequence to prime a polymerase chain reaction for             amplifying the aptamer sequence;         -   B is a single-stranded random nucleotide sequence from about             20 to about 110 nucleotides in length that contains specific             sequences binding to a first epitope of the target molecule;         -   D and R are a pair of complementary nucleotide sequences             from about 2 to about 20 nucleotides in length, wherein D             and R have a free energy for association from about 5.5             kcal/mole to about 8.0 kcal/mole at a temperature from             approximately 21° C. to about 40° C. and at a salt             concentration of approximately 1 mM to about 100 mM;         -   P is an epitope binding agent that binds to a second epitope             on the target molecule. The epitope binding agent will vary             depending upon the embodiment, but is selected from the             group comprising an aptamer, an antibody, an antibody             fragment, a double-stranded DNA sequence, a ligand, a ligand             fragment, a receptor, a receptor fragment, a polypeptide, a             peptide, a coenzyme, a coregulator, an allosteric molecule,             and an ion; and         -   Q is a flexible linker.

Generally speaking, A and C are each different DNA sequences ranging from about 7 to about 35 nucleotides in length and function as polymerase chain reaction primers to amplify the nucleic acid construct. In another embodiment, A and C range from about 15 to about 25 nucleotides in length. In yet another embodiment, A and C range from about 15 to about 20 nucleotides in length. In still another embodiment, A and C range from about 16 to about 18 nucleotides in length. In an exemplary embodiment, A and C are 18 nucleotides in length. Typically, A and C have an average GC content from about 53% to 63%. In another embodiment, A and C have an average GC content from about 55% to about 60%. In a preferred embodiment, A and C will have an average GC content of about 60%.

B is typically a single-stranded oligonucleotide synthesized by randomly selecting and inserting a nucleotide base (A, C, T, G in the case of DNA, or A, C, U, G in the case of RNA) at every position of the oligonucleotide. In one embodiment, B encodes an aptamer sequence that binds to the first epitope on the target. In another embodiment B is comprised of DNA bases. In yet another embodiment, B is comprised of RNA bases. In another embodiment, B is comprised of modified nucleic acid bases, such as modified DNA bases or modified RNA bases. Modifications may occur at, but are not restricted to, the sugar 2′ position, the C-5 position of pyrimidines, and the 8-position of purines. Examples of suitable modified DNA or RNA bases include 2′-fluoro nucleotides, 2′-amino nucleotides, 5′-aminoallyl-2′-fluoro nucleotides and phosphorothioate nucleotides (monothiophosphate and dithiophosphate). In a further embodiment, B is about 20 to 110 nucleotides in length. In another embodiment, B is from about 25 to about 75 nucleotides in length. In yet another embodiment, B is from about 30 to about 60 nucleotides in length.

In one embodiment, D and R are complementary nucleotide sequences from about 2 to about 20 nucleotides in length. In another embodiment, D and R are from about 4 to about 15 nucleotides in length. In a preferred embodiment, D and R are from about 5 to about 7 nucleotides in length. In one embodiment, D and R have a free energy for association from about 5.2 kcal/mole to about 8.2 kcal/mole as measured in the selection buffer conditions, defined below. In another embodiment, D and R have a free energy for association from about 6.0 kcal/mole to about 8.0 kcal/mole as measured in the selection buffer conditions defined below. In yet another embodiment, D and R have a free energy for association from about 7.0 kcal/mole to 8.0 kcal/mole in the selection buffer conditions. In a preferred embodiment, D and R have a free energy for association of 7.5 kcal/mole in the selection buffer conditions described below.

Q may be a nucleotide sequence from about 10 to about 100 nucleotides in length. In one embodiment, Q is from 10 to about 25 nucleotides in length. In another embodiment, Q is from about 25 to about 50 nucleotides in length. In a further embodiment, Q is from about 50 to about 75 nucleotides in length. In yet another embodiment, Q is from about 75 to about 100 nucleotides in length. In each embodiment, the nucleotides may be any of the nucleotide bases in DNA or RNA (A, C, T, G in the case of DNA, or A, C, U, G in the case of RNA). In one embodiment Q is comprised of DNA bases. In another embodiment, Q is comprised of RNA bases. In yet another embodiment, Q is comprised of modified nucleic acid bases, such as modified DNA bases or modified RNA bases. Modifications may occur at, but are not restricted to, the sugar 2′ position, the C-5 position of pyrimidines, and the 8-position of purines. Examples of suitable modified DNA or RNA bases include 2′-fluoro nucleotides, 2′-amino nucleotides, 5′-aminoallyl-2′-fluoro nucleotides and phosphorothioate nucleotides (monothiophosphate and dithiophosphate). In a further embodiment, B may comprise nucleotide mimics. Examples of nucleotide mimics include locked nucleic acids (LNA), peptide nucleic acids (PNA), and phosphorodiamidate morpholino oligomers (PMO). Alternatively, Q may be a polymer of bifunctional chemical linkers. In one embodiment the bifunctional chemical linker is heterobifunctional. Suitable heterobifunctional chemical linkers include sulfoSMCC (Sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate), and lc-SPDP (N-Succinimidyl-6-(3′-(2-PyridylDithio)-Propionamido)-hexanoate). In another embodiment the bifunctional chemical linker is homobifunctional. Suitable homobifunctional linkers include disuccinimidyl suberate, disuccinimidyl glutarate, and disuccinimidyl tartrate. Additional suitable linkers are illustrated in the Examples, such as the phosphoramidate form of Spacer 18 comprised of polyethylene glycol. In one embodiment, Q is from 0 to about 500 angstroms in length. In another embodiment, Q is from about 20 to about 400 angstroms in length. In yet another embodiment, Q is from about 50 to about 250 angstroms in length.

In a preferred embodiment, A and C are approximately 18 nucleotides in length and have an average GC content of about 60%; B is about 30 to about 60 nucleotides in length; Q is a linker comprising a nucleotide sequence that is from about 10 to 100 nucleotides in length or a bifunctional chemical linker; and D and R range from about 5 to about 7 nucleotides in length and have a free energy of association of about 7.5 kcal/mole.

As will be appreciated by those of skill in the art, the choice of epitope binding agent, P, can and will vary depending upon the particular target molecule. By way of example, when the target molecule is a protein P may be an aptamer, peptide, or antibody. By way of further example, when P is double stranded nucleic acid the target molecule is typically a macromolecule that binds to DNA or a DNA binding protein. Suitable epitope binding agents, depending upon the target molecule, include agents selected from the group consisting of an aptamer, an antibody, an antibody fragment, a double-stranded DNA sequence, a ligand, a ligand fragment, a receptor, a receptor fragment, a polypeptide, a peptide, a coenzyme, a coregulator, an allosteric molecule, and an ion. In an exemplary embodiment, P is an aptamer sequence ranging in length from about 20 to about 110 bases. In another embodiment, P is an antibody selected from the group consisting of polyclonal antibodies, ascites, Fab fragments, Fab′ fragments, monoclonal antibodies, and humanized antibodies. In a preferred embodiment, P is a monoclonal antibody. In an additional embodiment, P is a double stranded DNA.

Typically in the method, a plurality of nucleic acid constructs, A-B-C-D, are contacted with the epitope bind agent construct, P-Q-R, and the target molecule in the presence of a selection buffer to form a mixture. Several selection buffers are suitable for use in the invention. A suitable selection buffer is typically one that facilitates non-covalent binding of the nucleic acid construct to the target molecule in the presence of the epitope binding agent construct. In one embodiment, the selection buffer is a salt buffer with salt concentrations from about 1 mM to 100 mM. In another embodiment, the selection buffer is comprised of Tris-HCl, NaCl, KCl, and MgCl₂. In a preferred embodiment, the selection buffer is comprised of 50 mM Tris-HCl, 100 mM NaCl, 5 mM KCl, and 1 mM MgCl₂. In one embodiment, the selection buffer has a pH range from about 6.5 to about 8.5. In another embodiment, the selection buffer has a pH range from about 7.0 to 8.0. In a preferred embodiment, the pH is 7.5. Alternatively, the selection buffer may additionally contain analytes that assist binding of the constructs to the target molecule. Suitable examples of such analytes can include, but are not limited to, protein co-factors, DNA-binding proteins, scaffolding proteins, or divalent ions.

The mixture of the plurality of nucleic acid constructs, epitope binding agent constructs and target molecules are incubated in selection buffer from about 10 to about 45 min. In yet another embodiment, the incubation is performed for about 15 to about 30 min. Typically, the incubation is performed at a temperature range from about 21° C. to about 40° C. In another embodiment, the incubation is performed at a temperature range from about 20° C. to about 30° C. In yet another embodiment, the incubation is performed at 35° C. In a preferred embodiment, the incubation is performed at 25° C. for about 15 to about 30 min. Generally speaking after incubation, the mixture will typically comprise complexes of the target molecule having nucleic acid construct bound to a first epitope and epitope binding agent construct bound to a second epitope of the target molecule. The mixture will also comprise unbound nucleic acid constructs and epitope binding agent constructs.

The complex comprising the target molecule having bound nucleic acid construct and bound epitope binding agent construct is preferably isolated from the mixture. In one embodiment, nitrocellulose filters are used to separate the complex from the mixture. In an alternative embodiment magnetic beads are used to separate the complex from the mixture. In yet another embodiment sepharose beads can be used to separate the complex from the mixture. In an exemplary embodiment, strepavidin-linked magnetic beads are used to separate the complex from the mixture.

Optionally, the target molecules are subjected to denaturation and then the nucleic acid constructs purified from the complex. In one embodiment, urea is used to denature the target molecule. In a preferred embodiment, 7M urea in 1M NaCl is used to denature the target molecule. The nucleic acid constructs may be purified from the target molecule by precipitation. In another embodiment, the nucleic acid constructs are precipitated with ethanol. In yet another embodiment, the nucleic acid constructs are precipitated with isopropanol. In one embodiment, the precipitated DNA is resuspended in water. Alternatively, the precipitated DNA is resuspended in TE buffer.

Generally speaking, the purified, resuspended nucleic acid constructs are then amplified using the polymerase chain reaction (PCR). If the nucleic acid construct contains a B comprised of RNA bases, reverse transcriptase is preferably used to convert the RNA bases to DNA bases before initiation of the PCR. The PCR is performed with primers that recognize both the 3′ and the 5′ end of the nucleic acid constructs in accordance with methods generally known in the art. In one embodiment, either the 3′ or 5′ primer is attached to a fluorescent probe. In an alternative embodiment, either the 3′ or the 5′ primer is attached to fluorescein. In another embodiment, either the 3′ or 5′ primer is biotinylated. In a preferred embodiment, one primer is labeled with fluorescein, and the other primer is biotinylated.

In addition to primers, the PCR reaction contains buffer, deoxynucleotide triphosphates, polymerase, and template nucleic acid. In one embodiment, the PCR can be performed with a heat-stable polymerase. In a preferred embodiment, the concentrations of PCR reactants are outlined in the examples section as follows: 80 uL of dd H2O, 10 uL of 10×PCR buffer, 6 uL of MgCl₂, 0.8 uL 25 mM dNTPs, 1 uL 50 uM primer 1 (modified with fluorescein), 1 uL 50 uM primer 2 (biotinylated), 0.5 uL Taq polymerase, and 1 uL of template.

In another embodiment, the PCR consists of a warm-up period, where the temperature is held in a range between about 70° C. and about 74° C. Subsequently, the PCR consists of several cycles (about 8 to about 25) of 1) incubating the reaction at a temperature between about 92° C. and about 97° C. for about 20 sec to about 1 min; 2) incubating the reaction at a temperature between about 48° C. and about 56° C. for about 20 sec to about 1 min; and 3) incubating the reaction at a temperature between about 70° C. and about 74° C. for about 45 sec to about 2 min. After the final cycle, the PCR is concluded with an incubation between about 70° C. and about 74° C. for about 3 min to about 10 min. In an alternative embodiment, the reaction consists of 12-18 cycles. A preferred embodiment of the PCR, as outlined in the examples section, is as follows: 5 min at 95° C., sixteen cycles of 30 s at 95° C., 30 s at 50° C., and 1 min at 72° C., and then an extension period of 5 min at 72° C.

Typically after PCR amplification, the double-stranded DNA PCR product is separated from the remaining PCR reactants. One exemplary embodiment for such separation is subjecting the PCR product to agarose gel electrophoresis. In another embodiment, the PCR product is separated in a low melting point agarose gel. In a preferred embodiment, the gel is a native 10% acrylamide gel made in TBE buffer. In one embodiment, the band(s) having the double-stranded DNA PCR product are visualized in the gel by ethidium bromide staining. In another embodiment, the band(s) are visualized by fluorescein fluorescence. Irrespective of the embodiment, the bands are typically excised from the gel by methods generally known in the art.

Generally speaking, the double-stranded gel-purified PCR product is separated into single-stranded DNA in accordance with methods generally known in the art. One such embodiment involves using a basic pH to denature the double helix. In another embodiment, 0.15N NaOH is used to denature the helix. In still another embodiment, streptavidin linked beads are used to separate the denatured DNA strands. In a preferred embodiment, magnetic streptavidin beads are used to separate the denatured DNA strands.

The method of the invention typically involves several rounds of selection, separation, amplification and purification in accordance with the procedures described above until nucleic acid constructs having the desired binding affinity for the target molecule are selected. In accordance with the method, the single-stranded DNA of estimated concentration is used for the next round of selection. In one embodiment, the cycle of selection, separation, amplification, purification, and strand separation is performed From about 4 to about 20 times. In another embodiment, the said cycle is performed from about 12 to about 18 times. In yet another embodiment, the said cycle is performed until the measured binding-activity of the selected nucleic acid constructs reaches the desired strength.

Alternatively, the single DNA strand attached to the strepavidin-linked beads is used as a template for RNA polymerase. In this embodiment, after the RNA polymerase is finished, the supernatant contains the RNA nucleic acid construct that can be used in another round of RNA aptamer selection.

In an alternative method, if a RNA aptamer is being selected, the double-stranded, gel-purified PCR DNA product is transcribed with RNA polymerase to produce a single-stranded RNA construct. In such a case, A will typically contain a sequence encoding a promoter recognized by RNA polymerase. In one embodiment, double-stranded, gel-purified PCR DNA product attached to strepavidin-linked beads is used as a template for RNA polymerase. In this embodiment, after the RNA polymerase reaction, the supernatant containing the RNA nucleic acid construct can be used in another round of RNA aptamer selection.

Generally speaking, after the nucleic acid constructs have reached the desired binding specificity, the nucleic acid constructs are cloned, and the cloned DNA is sequenced. In one embodiment, the sequences are used in aptamer constructs either alone or as part of a molecular biosensor.

(B) Method for Simultaneous Selection of Two or More Aptamers

Another aspect of the invention is a method for simultaneously selecting two or more aptamers. The aptamers selected by the method each bind to the same target molecule at two distinct epitopes. Typically, the method comprises contacting a plurality of pairs of nucleic acid constructs with a target molecule to form a mixture. The mixture will generally comprise complexes having target molecule bound with a pair of nucleic acid constructs at distinct epitope sites. According to the method, the complex is isolated from the mixture and the nucleic acid constructs are purified from the complex. The aptamers selected by the method of the invention will comprise the pair of purified nucleic acid constructs.

In the method of the invention, the first nucleic acid constructs comprises: A-B-C-D

The second nucleic acid construct comprises: E-F-G-H.

-   -   wherein:         -   A, C, E, and G are each different DNA sequences from about             10 to about 30 nucleotides in length, A and C together             comprising a sequence to prime a polymerase chain reaction             for amplifying a first aptamer sequence, and E and G             together comprising a sequence to prime a polymerase chain             reaction for amplifying a second aptamer sequence;         -   B is a single-stranded random nucleotide sequence from about             20 to about 110 nucleotides in length that contains specific             sequences binding to a first epitope of the target molecule;         -   D and H are a pair of complementary nucleotide sequences             from about 2 to about 20 nucleotides in length, wherein D             and H have a free energy for association from about 5.5             kcal/mole to about 8.0 kcal/mole at a temperature from             approximately 21° C. to about 40° C. and at a salt             concentration of approximately 1 mM to about 100 mM; and         -   F is a single-stranded random nucleotide sequence from about             20 to about 110 nucleotides in length that contains specific             sequences binding to the second epitope of the target             molecule.

In another embodiment, A, C, E and G are each different DNA sequences ranging from about 7 to about 35 nucleotides in length. In another embodiment, A, C, E, and G range from about 15 to about 25 nucleotides in length. In yet another embodiment, A, C, E, and G range from about 15 to about 20 nucleotides in length. In still another embodiment, A, C, E and G range from about 16 to about 18 nucleotides in length. In an exemplary embodiment, A, C, E and G are 18 nucleotides in length. Generally speaking, A, C, E and G have an average GC content from about 53% to 63%. In another embodiment, A, C, E and G have an average GC content from about 55% to about 60%. In a preferred embodiment, A, C, E and G will have an average GC content of about 60%.

In one embodiment, B and F are single-stranded oligonucleotides synthesized by randomly selecting and inserting a nucleotide base (A, C, T, G in the case of DNA, or A, C, U, G in the case of RNA) at every position of the oligonucleotide. In a preferred embodiment, B and F encode an aptamer sequence, such that B binds to the first epitope on the target molecule and F binds to the second epitope on the target molecule. In one embodiment B and F are comprised of DNA bases. In another embodiment, B and F are comprised of RNA bases. In yet another embodiment, B and F are comprised of modified nucleic acid bases, such as modified DNA bases. Modifications may occur at, but are not restricted to, the sugar 2′ position, the C-5 position of pyrimidines, and the 8-position of purines. Examples of suitable modified DNA or RNA bases include 2′-fluoro nucleotides, 2′-amino nucleotides, 5′-aminoallyl-2′-fluoro nucleotides and phosphorothioate nucleotides (monothiophosphate and dithiophosphate). In typical embodiments, B and F are about 20 to 110 nucleotides in length. In another embodiment, B and F are from about 25 to about 75 nucleotides in length. In yet another embodiment, B and F are from about 30 to about 60 nucleotides in length.

D and H are complementary nucleotide sequences from about 2 to about 20 nucleotides in length. In another embodiment, D and H are from about 4 to about 15 nucleotides in length. In a preferred embodiment, D and H are from about 5 to about 7 nucleotides in length. In one embodiment, D and H have a free energy for association from about 5.2 kcal/mole to about 8.2 kcal/mole as measured in the selection buffer conditions, defined below. In another embodiment, D and H have a free energy for association from about 6.0 kcal/mole to about 8.0 kcal/mole as measured in the selection buffer conditions. In yet another embodiment, D and H have a free energy for association from about 7.0 kcal/mole to 8.0 kcal/mole in the selection buffer conditions. In a preferred embodiment, D and H have a free energy for association of 7.5 kcal/mole in the selection buffer conditions.

In a preferred embodiment, A, C, E and G are approximately 18 nucleotides in length and have an average GC content of about 60%, B and F are about 30 to about 60 nucleotides in length, and D and H range from about 5 to about 7 nucleotides in length and have a free energy of association of about 7.5 kcal/mole.

The method for simultaneous selection is initiated by contacting a plurality of pairs of the nucleic acid constructs A-B-C-D and E-F-G-H with the target molecule in the presence of a selection buffer to form a complex. Generally speaking, suitable selection buffers allow non-covalent simultaneous binding of the nucleic acid constructs to the target molecule. The method for simultaneous selection then involves the same steps of selection, separation, amplification and purification as described in section (a) above involving methods for the selection of an aptamer in the presence of an epitope binding agent construct, with the exception that the PCR is designed to amplify both nucleic acid constructs (A-B-C-D and E-F-G-H), using primers to A, C, E, and G. Typically several rounds of selection are performed until pairs of nucleic acid constructs having the desired affinity for the target molecule are selected. In one embodiment, the cycle of selection, separation, amplification, purification, and strand separation is performed from about 4 to about 20 times. In another embodiment, the cycle is performed from about 12 to about 18 times.

After the pair of nucleic acid constructs has reached the desired binding specificity, the nucleic acid constructs are cloned, and the cloned DNA is sequenced. The resulting nucleic acid constructs comprise a first aptamer that binds to a first epitope on the target molecule and a second aptamer that binds to a second epitope on the target molecule.

In another aspect of the invention, two aptamers can be simultaneously selected in the presence of a bridging construct comprised of S-T-U. In one embodiment, S and U are complementary nucleotide sequences from about 2 to about 20 nucleotides in length. In another embodiment, S and U are from about 4 to about 15 nucleotides in length. In a preferred embodiment, S and U are from about 5 to about 7 nucleotides in length. In one embodiment, S and U have a free energy for association from about 5.2 kcal/mole to about 8.2 kcal/mole as measured in the selection buffer conditions, defined below. In another embodiment, S and U have a free energy for association from about 6.0 kcal/mole to about 8.0 kcal/mole as measured in the selection buffer conditions. In yet another embodiment, S and U have a free energy for association from about 7.0 kcal/mole to 8.0 kcal/mole in the selection buffer conditions. In a preferred embodiment, S and U have a free energy for association of 7.5 kcal/mole in the selection buffer conditions.

T may be a nucleotide sequence from about 10 to about 100 nucleotides in length. In one embodiment, T is from 10 to about 25 nucleotides in length. In another embodiment, T is from about 25 to about 50 nucleotides in length. In a further embodiment, T is from about 50 to about 75 nucleotides in length. In yet another embodiment, T is from about 75 to about 100 nucleotides in length. In each embodiment, the nucleotides may be any of the nucleotide bases in DNA or RNA (A, C, T, G in the case of DNA, or A, C, U, G in the case of RNA). In one embodiment T is comprised of DNA bases. In another embodiment, T is comprised of RNA bases. In yet another embodiment, T is comprised of modified nucleic acid bases, such as modified DNA bases or modified RNA bases. Modifications may occur at, but are not restricted to, the sugar 2′ position, the C-5 position of pyrimidines, and the 8-position of purines. Examples of suitable modified DNA or RNA bases include 2′-fluoro nucleotides, 2′-amino nucleotides, 5′-aminoallyl-2′-fluoro nucleotides and phosphorothioate nucleotides (monothiophosphate and dithiophosphate). In a further embodiment, B may comprise nucleotide mimics. Examples of nucleotide mimics include locked nucleic acids (LNA), peptide nucleic acids (PNA), and phosphorodiamidate morpholino oligomers (PMO). Alternatively, T may be a polymer of bifunctional chemical linkers. In one embodiment the bifunctional chemical linker is heterobifunctional. Suitable heterobifunctional chemical linkers include sulfoSMCC (Sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate), and lc-SPDP (N-Succinimidyl-6-(3′-(2-PyridylDithio)-Propionamido)-hexanoate). In another embodiment the bifunctional chemical linker is homobifunctional. Suitable homobifunctional linkers include disuccinimidyl suberate, disuccinimidyl glutarate, and disuccinimidyl tartrate. Additional suitable linkers are illustrated in the Examples, such as the phosphoramidate form of Spacer 18 comprised of polyethylene glycol. In one embodiment, T is from 0 to about 500 angstroms in length. In another embodiment, T is from about 20 to about 400 angstroms in length. In yet another embodiment, T is from about 50 to about 250 angstroms in length.

In one embodiment, S is complementary to D and U is complementary to H. In another embodiment, S and U will not bind to D and H unless S, U, D, and H are brought in close proximity by the A-B-C-D construct and the E-F-G-H construct binding to the target.

In this embodiment of the invention utilizing the bridging construct, the method is initiated in the presence of nucleic acid constructs A-B-C-D and E-F-G-H, and the bridging construct S-T-U. Generally speaking, the method is performed as described with the same steps detailed above. In one embodiment, after the final round of selection, but before cloning, the bridging construct is ligated to the A-B-C-D construct and the E-F-G-H construct. This embodiment allows the analysis of pairs of selected nucleic acid sequences that are best suited for use in a molecular biosensor.

Definitions

As used herein, the term “analyte” refers generally to a ligand, chemical moiety, compound, ion, salt, metal, enzyme, secondary messenger of a cellular signal transduction pathway, drug, nanoparticle, environmental contaminant, toxin, fatty acid, steroid, hormone, carbohydrate, amino acid, peptide, polypeptide, protein or other amino acid polymer, microbe, virus or any other agent which is capable of binding to a polypeptide, protein or macromolecular complex in such a way as to create an epitope or alter the availability of an epitope for binding to an aptamer.

The term “aptamer” refers to a polynucleotide, generally a RNA, modified RNA, DNA, or modified DNA that has a useful biological activity in terms of biochemical activity, molecular recognition or binding attributes. Usually, an aptamer has a molecular activity such as binding to a target molecule at a specific epitope (region).

The term “epitope” refers generally to a particular region of a target molecule. Examples include an antigen, a hapten, a molecule, a polymer, a prion, a microbe, a cell, a peptide, polypeptide, protein, or macromolecular complex. An epitope may consist of a small peptide derived from a larger polypeptide. An epitope may be a two or three-dimensional surface or surface feature of a polypeptide, protein or macromolecular complex that comprises several non-contiguous peptide stretches or amino acid groups.

The term “epitope binding agent” refers to a substance that is capable of binding to a specific epitope of an antigen, a polypeptide, a protein or a macromolecular complex. Non-limiting examples of epitope binding agents include aptamers, thioaptamers, double-stranded DNA sequence, peptides and polypeptides, ligands and fragments of ligands, receptors and fragments of receptors, antibodies and fragments of antibodies, polynucleotides, coenzymes, coregulators, allosteric molecules, peptide nucleic acids, locked nucleic acids, phosphorodiamidate morpholino oligomers (PMO), and ions.

The term “epitope binding agent construct” refers to a construct that contains an epitope-binding agent and can serve in a “molecular biosensor” with another epitope binding agent construct. Preferably, an epitope binding agent construct also contains a “linker,” and a “signaling oligo”. Epitope binding agent constructs can be used to initiate the aptamer selection methods of the invention. An aptamer construct is a special kind of epitope binding agent construct wherein the epitope binding agent is an aptamer.

The term “molecular biosensor” refers to a construct comprised of at least two epitope binding agent constructs. The molecular biosensor can be used for detecting or quantifying the presence of a target molecule.

The term “nucleic acid construct” refers to a molecule comprising a random nucleic acid sequence flanked by two primers. Preferably, a nucleic acid construct also contains a signaling oligo. Nucleic acid constructs are used to initiate the aptamer selection methods of the invention.

The term “signaling oligo” means a short (generally 2 to 15 nucleotides, preferably 5 to 7 nucleotides in length) single-stranded polynucleotide. Signaling oligos are typically used in pairs comprising a first signaling oligo and a second signaling oligo. Preferably, the first signaling oligo sequence is complementary to the second signaling oligo. Preferably, the first signaling oligo and the second signaling oligo can not form a stable association with each other through hydrogen bonding unless the first and second signaling oligos are brought into close proximity to each other through the mediation of a third party agent.

As used herein, the term “linker” or “linker molecule” refers to a polymer attached to an epitope binding agent construct. The attachment may be covalent or non-covalent. It is envisioned that the linker can be a polymer of amino acids or nucleotides. A preferred linker molecule is flexible and does not interfere with the binding of a nucleic acid binding factor to the set of nucleic acid components.

As used herein, the term “macromolecular complex” refers to a composition of matter comprising a macromolecule. Preferably, these are complexes of one or more macromolecules, such as polypeptides, lipids, carbohydrates, nucleic acids, natural or artificial polymers and the like, in association with each other. The association may involve covalent or non-covalent interactions between components of the macromolecular complex. Macromolecular complexes may be relatively simple, such as a ligand bound polypeptide, relatively complex, such as a lipid raft, or very complex, such as a cell surface, virus, bacteria, spore and the like. Macromolecular complexes may be biological or non-biological in nature.

As various changes could be made in the methods described herein without departing from the scope of the invention, it is intended that all matter contained in the above description and in the examples given below, shall be interpreted as illustrative and not in a limiting sense.

EXAMPLES

The following examples illustrate the invention.

Example 1 Methods for Selecting Aptamers for use in Molecular Sensors

FIG. 2 summarizes five possible methods for selecting aptamers useful in the practice of the invention. Panel A depicts the selection of an additional aptamer in the presence of a target bound to a known aptamer. The nucleic acid construct is comprised of a signaling oligo, represented by the hatched bar, and two primers flanking a random DNA sequence. In practice, the signaling oligo is treated as a specific subpart of the primer in the nucleic acid construct. A complimentary signaling oligo is attached to the pre-selected aptamer via a long flexible linker. Here, the process begins by combining the nucleic acid construct, the target, and the known aptamer construct. Selection of aptamers using such a random sequence construct will be biased towards aptamers capable of binding to the target at an epitope distinct from the epitope of the known aptamer construct, and that will function in molecular biosensors depicted in FIG. 3A.

An alternative scenario is depicted in panel B, which describes the simultaneous selection of two aptamers binding two distinct epitopes of the target. The nucleic acid constructs are comprised of signaling oligos (represented by the hatched bars at the end of primer 1 and primer 4) and two primers flanking either side of a random-sequence. There are at least two different types of nucleic acid constructs, each type containing unique primer sequences. In panel B, one type contains primers 1 and 2, and the second contains primers 3 and 4. In this example, the process begins with combining both types of nucleic acid constructs, and the target. Selection of aptamers using such random sequence constructs will be biased towards aptamers capable of binding to the target simultaneously at two distinct epitopes of the protein, and that will function in sensors depicted in FIG. 3A.

Panel C depicts an alternative design for simultaneous selection of two aptamers binding two distinct epitopes of the target. In addition to the two different types of nucleic acid constructs, a third bridging construct is used. The bridging construct comprises an additional pair of short oligonucleotides (hatched bars) connected by a flexible linker. These oligonucleotides will be complementary to the short oligonucleotides at the end of the nucleic acid constructs. The presence of the bridging construct during selection will provide a bias towards selecting pairs of aptamers capable of simultaneously binding the target. Before cloning of the selected aptamers (after the last selection) the pairs of selected sequences will be enzymatically ligated using T4 ligase to preserve the information regarding the preferred pairs between various selected aptamers.

In a fourth alternate embodiment, a second aptamer can be selected in the presence of a target bound by an antibody (FIG. 2D). The signaling oligo in the nucleic acid construct, depicted by the hatched bar, is complementary to the signaling oligo attached to the antibody via a long flexible linker. The process begins by combining the nucleic acid construct, the target, and the antibody construct. Selection of an aptamer using such a random sequence construct will be biased towards aptamers able to bind to the protein at an epitope distinct from the antibody epitope and will function in sensors depicted in FIG. 3C.

In a fifth alternate embodiment, a second aptamer can be selected in the presence of the target bound to a double-stranded DNA fragment (FIG. 2E). The signaling oligo in the nucleic acid construct, depicted by the hatched bar, is complementary to the signaling oligo attached to the double-stranded DNA construct via a long flexible linker. The process begins by combining the nucleic acid construct, the target, and the double-stranded DNA construct. Selection of an aptamer using such a random sequence construct will be biased towards aptamers able to bind to the target at a site distinct from the double-stranded DNA binding site and will function in sensors depicted in FIG. 3B.

Example 2 Sequential Selection of Aptamers that Bind to Thrombin for use in Making a Thrombin Sensor

The results of selecting an aptamer in the presence of a known aptamer construct are depicted in FIG. 5. The in vitro evolution procedure was initiated using a selection construct containing a 33 nt random sequence (THR11)(Table 1) in the presence of the known THR22 aptamer construct (THR22)(Table 1) and the target, in this case thrombin (FIG. 5A). Five μM of THR11 were added to 5 μM THR22 in a total of 1 mL of buffer (50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 5 mM KCl and 1 mM MgCl₂). The mixture was boiled for approximately 1 min, and allowed to cool to room temperature (RT). Thrombin (200 nM) was added, and the mixture was incubated for 15-30 min at RT. Next, the mixture was spun through a nitrocellulose filter (NCF), followed by 2 washes of 1 mL and a single wash of 0.5 mL. Pre-warmed urea (200 μL of 7M solution containing 1M NaCl) was loaded on the NCF, and incubated for 15 min at 37° C. The DNA/urea mixture was eluted, and the DNA was precipitated with ethanol (added cold ethanol (2.5× the volume of eluted DNA) and incubated for at least two hours at −20° C.). The precipitated DNA was centrifuged, the supernatant removed, and the subsequent pellet was dried in a speed-vac. The pellet was re-dissolved in 20 μL of water, and used as a template for the PCR reaction.

Each PCR reaction contained 80 μL of dd H2O, 10 μL of 10×PCR buffer, 6 μL of MgCl₂, 0.8 μL 25 mM dNTPs, 1 μL 50 uM primer 1 (modified with fluorescein), 1 uL 50 μM primer 2 (biotinylated), 0.5 μL Taq polymerase, and 1 μL of template. The reaction cycle consisted of 5 min at 95° C., sixteen cycles of 30 s at 95° C., 30 s at 50° C., and 1 min at 72° C., and 5 min at 72° C. The pooled samples were allowed to cool, and subsequently separated on a polyacrylamide gel. The band(s) of interest were visualized by utilizing the fluorescein tag, and were excised from the gel. The gel pieces were transferred to a microtube and crushed using a pipet tip. The gel pieces were covered with diffusion buffer (100 mM Tris (pH 8.0), 0.5 M NaCl, 5 mM EDTA) and the mixture was incubated for at least two hours at 50° C. After centrifugation the supernatant was filtered through an empty Bio-Rad microspin column. The gel pieces were washed with fresh diffusion buffer, and the process repeated for a second time. The supernatants from the first and second procedures were combined.

Pre-equilabrated (1 M NaCl, 50 mM Tris (pH 8.0), and 1 mM EDTA) DYNAL magnetic streptavidin beads were mixed with the gel-purified DNA, and incubated at RT for 30 min with constant shaking. The supernatant was removed, and the beads were washed once with 500 μL, once with 250 μL, and once with 100 μL of buffer. Next, the beads were incubated for 30 min at 37° C. with 50 μL of 0.15N NaOH. The supernatant containing the fluorescein labeled DNA was removed and filtered through a G-25 Sephadex microspin column pre-equilibrated with buffer. The estimated concentration of the recovered DNA was calculated by comparison to a known amount of fluorescein-labeled primer.

The second round of selection began by combining 50 nM of the recovered DNA and 50-1000 nM of THR22 in a total of 50 μL of selection buffer. The DNA mixture was boiled for 1 min, and allowed to cool to RT. Subsequently, the DNA mixture was filtered through a pre-equilibrated NCF to remove DNA sequences with affinity for the NCF. Thrombin (20 nM) was added to the filtered DNA and the mixture was incubated for 15-10 min at RT. Next, the mixture was spun through another pre-equilibrated NCF, followed by two washes of 100 μL. After incubation with 100 μL of urea (7M in a buffer of 1 M NaCl) for 15 min at 37° C., the DNA-thrombin complexes were eluted from the NCF. The DNA in the eluted solution was precipitated with alcohol (see above) and re-suspended in 20 μL of water. This was used as a template for the PCR reaction. PCR products were purified by electrophoresis on polyacrylamide gel and the single-stranded DNA was obtained from purified PCR products as described above for the first selection. Subsequent selections were repeated until the detected thrombin-binding activity reached a maximum (FIG. 5B).

Panel B depicts the thrombin-binding activity of single-stranded DNAs obtained after each indicated round of selection. Measurable thrombin-binding activity appeared after the 4th selection and reached maximum binding activity after the 12th selection. Binding was measured in the presence of excess THR22. DNA obtained after the 12th selection was cloned and the DNA from individual clones was sequenced. Panel C depicts the sequence alignment (using ClustalX) of the individual clones. Clones obtained from 4 independent selection experiments are shown. These selections were performed using the following pairs of aptamer constructs and selection constructs: THR22 and THR 11; THR25 and THR 11; THR42 and THR11; THR43 and THR 11 (Table 1). Several families of highly conserved sequences are easily visible in panel C.

One sequence, which appeared in 4 clones (clones 20, 21, 24, and 26) shown in FIG. 5, was used to create a functional thrombin biosensor (FIG. 6). The cloned sequence was used in two separate aptamer constructs. Each construct (THR 35 and THR 36) was comprised of the aptamer sequence, a linker, and a fluorescein-labeled signaling oligo. As shown in FIG. 6, THR 35 and THR 36 differ in the nucleotides flanking the cloned sequence. The other half of the thrombin biosensor (THR27, Table 1) contained the same aptamer sequence as the THR22 aptamer construct used in the selection, a linker, and a Texas Red-labeled complementary signaling oligo. Panels A and B depict the fluorescence image (sensitized acceptor emission) of microtiterplate wells containing 20 nM (panel A) or 100 nM (panel B) of the indicated thrombin sensor and the indicated concentrations of thrombin. For comparison, a sensor comprising aptamer constructs containing previously described thrombin aptamers (THR21 and THR27, Table 1) is shown.

Example 3 Simultaneous Selection of Aptamers that Bind to Thrombin

FIG. 7 depicts the results of simultaneously selecting two aptamers that bind to a target at distinct epitopes. The selection procedure began with two types of selection constructs, each containing a 30 nt random sequence (THR49 and THR50) (Table 1), and the target thrombin (panel A). The process proceeds as outlined above, with the exception that two sets of PCR reactions were performed, corresponding to the two different types of nucleic acid constructs initially used. The thrombin-binding activity of the mixture of single-stranded DNAs obtained after each indicated round of selection is shown in panel B. Measurable thrombin-binding activity appeared after the 6th selection and reached a maximum after the 14th selection. DNA obtained after the 14th selection was cloned and the DNA from individual clones was sequenced. Panel C depicts the sequence alignment (using ClustalX) of the clones. Several families of highly conserved sequences are easily visible.

Example 4 Selection of an Aptamer for use in a CRP Sensor with Double-Stranded DNA

FIG. 8 depicts the selection of an aptamer in the presence of a double-stranded DNA molecular recognition construct. Aptamers to the cAMP binding protein (“CRP”) were selected to bind at sites distinct from the DNA-binding site of the protein. Selection was initiated with a nucleic acid construct containing a 33 nt random sequence (MIS12)(Table 1). Additionally, a double-stranded DNA construct comprised of the CRP DNA binding site sequence (MIS10X3 hybridized with MIS11), a linker, and a signaling oligo was used for the process. (FIG. 8A). The selection process was essentially the same as outlined above for the thrombin aptamer. The only difference was that the buffer used in the selection contained 200 μL c-AMP, which is required for the double-stranded DNA construct to bind to CRP. In the first selection ˜5 uM MIS12 was combined with 400 nM MIS10X3/MIS11, 400 nM CRP, and 0.2 mM c-AMP. In subsequent selections 100 nM MIS10X3/MIS11, 50 nM selected single-stranded DNA constructs and 100 nM CRP were combined.

CRP binding activity of single-stranded DNA obtained after the indicated round of selection is depicted in FIG. 8B. Measurable CRP binding activity appeared after the 6th selection and reached a maximum after the 12th selection. Binding was measured in the presence of excess MIS10X3/MIS11. DNA obtained after the 12th selection was cloned and the DNA obtained from individual clones was sequenced. The sequence alignment (using ClustalX) of the clones is depicted in panel C. A conserved core sequence of ˜16 nucleotides could be identified.

TABLE 1 Sequence Construct Identifier Sequence Description THR11 CTG TCG TTA GTG AAG SEQ ID NO: 1 Construct containing 33 nt GTT NNN NNN NNN NNN random DNA sequence for NNN NNN NNN NNN NNN thrombin aptamer selection NNN NNN AAC GCC ATA TCA CAG ACG THR22 GGT TGG TGT GGT TGG SEQ ID NO: 2 Co-aptamer for thrombin (Spacer18)₂ GA CAG aptamer selection THR25 GGT TGG TGT GGT TGG SEQ ID NO: 3 Co-aptamer for thrombin (Spacer18)₅ AC GA CAG aptamer selection THR42 GGT TGG TGT GGT TGG SEQ ID NO: 4 co-aptamer for thrombin (Spacer18)₅ AAC GAC AG aptamer selection THR43 CTG TCG TT (Spacer18)₅ SEQ ID NO: 5 Construct containing 33 nt TTGAGTCAGCGTCGAGCA random DNA sequence for NNN NNN NNN NNN NNN thrombin aptamer selection NNN NNN NNN NNN NNN NNN TTC ACT GTG CTG CGG CTA THR27 GGT TGG TGT GGT TGG SEQ ID NO: 6 G15D aptamer connected via (Spacer18)₅ (C6 amino-dT) 5 Spacer18 linkers to 7 nt C GCA TCT “signaling” oligonucleotide containing amino-dT (near its 5′ end) THR35 5′ fluorescein AGA TGC G SEQ ID NO: 7 Thrombin sensor component (Spacer18)₅ AG GTT GGG GGT ACT AGG TAT CAA TGG GTA GGG TGG TGT AAG GC THR36 5′ fluorescein AGA TGC G SEQ ID NO: 8 Thrombin sensor component (Spacer18)₅ A GTG AAG GTT GGG GGT ACT AGG TAT CAA TGG GTA GGG TGG TGT AAC GCC ATA T THR21 5′ fluorescein AGA TGC G SEQ ID NO: 9 7 nt “signaling” (Spacer18)₅ AGT CCG TGG oligonucleotide labeled at 5′ TAG GGC AGG TTG GGG with fluorescein connected to TGA CT 60-18 [29] aptamer via 5 Spacer18 linkers THR49 CACCTGATCGCTCCTCGT SEQ ID NO: 10 Construct containing 30 nt NNN NNN NNN NNN NNN random DNA sequence for NNN NNN NNN NNN NNN simultaneous selection of CAG GAT GCA CAG GCA two thrombin aptamers CAA THR50 AGCCGCCATTCCATAGTG SEQ ID NO: 11 Construct containing 30 nt NNN NNN NNN NNN NNN random DNA sequence for NNN NNN NNN NNN NNN simultaneous selection of CAG GAT GCC GAT CAG two thrombin aptamers GTG MIS12 AGCCA T CTA ACT ATT SEQ ID NO: 12 Construct containing 33 nt CCC NNN NNN NNN NNN random DNA sequence for NNN NNN NNN NNN NNN CRP aptamer selection NNN NNN GAG CGA GAA ATT CTA GGT MIS10X3 AAC GCA ATA AAT GTG SEQ ID NO: 13 co-aptamer for CRP aptamer AAG TAG ATC ACA TTT selection TAG GCA CC (Spacer18)₅ GA TGGCT MIS11 GGT GCC TAA AAT GTG SEQ ID NO: 14 Complement to MIS10X3 ATC TAC TTC ACA TTT ATT GCG TT Clone 1 GGCGGTATGG SEQ ID NO: 15 Clone from FIG. 5 GCATAGCGTA ATGGGAGGTT GGT Clone 2 GGATGCGTAA SEQ ID NO: 16 Clone from FIG. 5 TGGTTAGGGT GGGTAGGGTA TCC Clone 3 GGATGCGTAA SEQ ID NO: 17 Clone from FIG. 5 TGGTTAGGGT GGGTAGGGTA TCC Clone 4 GGATGCGTAA SEQ ID NO: 18 Clone from FIG. 5 TGGTTAGGGT GGGTAGGGTA TCC Clone 5 GCAGTAGGTA SEQ ID NO: 19 Clone from FIG. 5 CTATATTGGC TAGGGTGGTC TGC Clone 6 GCAGTAGGTA SEQ ID NO: 20 Clone from FIG. 5 CTATATTGGC TAGGGTGGTC TGC Clone 7 GGCGGTATGG SEQ ID NO: 21 Clone from FIG. 5 GCATAGCGTA ATGGGAGGTC TGC Clone 8 GGATGCGTAA SEQ ID NO: 22 Clone from FIG. 5 TGGTTAGGGT GGGTAGGGTA TCC Clone 9 GGCGGTATGG SEQ ID NO: 23 Clone from FIG. 5 GTATAGCGTA ATGGGAGGTT GGT Clone 10 GGGGGTACTA SEQ ID NO: 24 Clone from FIG. 5 GGTATTAATG GGTAGGGTGG TGT Clone 11 CAGCAGGGAA SEQ ID NO: 25 Clone from FIG. 5 CGGAACGGTT AGGGTGGGTA GGG Clone 12 GCGGNGATAG SEQ ID NO: 26 Clone from FIG. 5 GTCGCGTAAG TTGGGTAGGG TGG Clone 13 CAGGATGGGT SEQ ID NO: 27 Clone from FIG. 5 AGGGTGGTCA GCGAAGCAGT AGG Clone 14 CAACGGTTGG SEQ ID NO: 28 Clone from FIG. 5 GTGAACTGTA GTGGCTTGGG GTG Clone 15 CAGGATGGGT SEQ ID NO: 29 Clone from FIG. 5 AGGGTGGTCA GCGAAGCAGT AGG Clone 16 CAGGATGGGT SEQ ID NO: 30 Clone from FIG. 5 AGGGTGGTCA GCGAAGCAGT AG Clone 17 GGCGAGAGCA SEQ ID NO: 31 Clone from FIG. 5 GCGTGATAGG GTGGGTAGGG TGG Clone 18 CAGGGTCAGG SEQ ID NO: 32 Clone from FIG. 5 GCTAGATGAT GCGATTAACC ATG Clone 19 CAGGATGGGT SEQ ID NO: 33 Clone from FIG. 5 AGGGTGGTCA GCGAAGCAGT AGG Clone 20 GGGGGTACTA SEQ ID NO: 34 Clone from FIG. 5 GGTATCAATG GGTAGGGTGG TGT Clone 21 GGGGGTACTA SEQ ID NO: 35 Clone from FIG. 5 GGTATCAATG GGTAGGGTGG TGT Clone 22 GGAGACGTAA SEQ ID NO: 36 Clone from FIG. 5 TGGGTTGGTT GGGAAGNGGA TCC Clone 23 GCATACGTAA SEQ ID NO:37 Clone from FIG. 5 TGGTCCGGTT GGGGCGGGTA TGT Clone 24 GGGGGTACTA SEQ iD NO: 38 Clone from FIG. 5 GGTATCAATG GGTAGGGTGG TGT Clone 25 GAGGGGACTT SEQ ID NO: 39 Clone from FIG. 5 AGGATGGGTA GGGTGGTAGG CCC Clone 26 GGGGGTACTA SEQ ID NO: 40 Clone from FIG. 5 GGTATCAATG GGTAGGGTGG TGT Clone 27 GGTCGGGGCA SEQ ID NO: 41 Clone from FIG. 5 TAGTAATGCT GGATTGGGCA GCT Clone 1-3 GGCACAACCC SEQ ID NO: 52 Clone from FIG. 7 GATATGGCTA TGAATCTGCC Clone 1-4 GGGTAGGGTG SEQ ID NO: 53 Clone from FIG. 7 GTTGTAATAG GGATTGCGAT Clone 1-5 GGGTAGGGTG SEQ ID NO: 54 Clone from FIG. 7 GTTGTAATAG GGATTGCGAT Clone 1-6 GGTGTGGGTG SEQ ID NO: 55 Clone from FIG. 7 GTTATTGGTG TAGAGCGGGT Clone 1-7 AATGGGGAGG SEQ ID NO: 56 Clone from FIG. 7 TTGGGGTGCG GGAGAGTGGT Clone 1-8 ACGCGTAGGA SEQ ID NO: 57 Clone from FIG. 7 TGGGTAGGGT GGTCGCGTTA Clone 1-9 GGGTAGGGTG SEQ ID NO: 58 Clone from FIG. 7 GTTGTAATAG GGATTGCGAT Clone 1-10 GGGCGAAGGT SEQ ID NO: 59 Clone from FIG. 7 ACGAAGACGG ATGCACGTGC Clone 2-1 AAGGCCGCCA SEQ ID NO: 60 Clone from FIG. 7 TCTGGGTCCG ACGACTACCA Clone 2-2 TAGGGTGGGT SEQ ID NO: 61 Clone from FIG. 7 AGGGTGGTCA ACTATGGGGG Clone 28 GGGTAGGAGC SEQ ID NO: 42 Clone from FIG. 5 AGTACACGCT GGAATGGGTC ACT Clone 29 GCAGTAGGTA SEQ ID NO: 43 Clone from FIG. 5 CTATATTGGC TAGGGTGGTC TGC Clone 30 GGGTAGGGTG SEQ ID NO: 44 Clone from FIG. 5 ACAGGGAGGA CGGAATGGGC ACT Clone 31 GCAGTAGGTA SEQ ID NO: 45 Clone from FIG. 5 CTATATTGGC TAGGGTGGTC TGC Clone 32 GCAGTAGGTA SEQ ID NO: 46 Clone from FIG. 5 CTATATTGGC TAGGGTGGTC TGC Clone 33 GCAGTAGGTA SEQ ID NO: 47 Clone from FIG. 5 CTATATTGGC TAGGGTGGTC TGC Clone 34 GGGGGTGCTA SEQ ID NO: 48 Clone from FIG. 5 GGTATTAAAG GGTAGGGTGG TGT Clone 35 GCAGTAGGTA SEQ ID NO: 49 Clone from FIG. 5 CTATGTCGGG TCGGGTGGTC TGC Clone 1-1 GGGTAGGGTG SEQ ID NO: 50 Clone from FIG. 7 GTTGTAATAG GGATTGCGAT Clone 1-2 GGGTAGGGTG SEQ ID NO: 51 Clone from FIG. 7 GTTGTAATAG GGATTGCGAT Clone 2-3 GGGTGGCTGG SEQ ID NO: 62 Clone from FIG. 7 TCAAGGAGAT AGTACGATGC Clone 2-4 GGTAGGGTGG SEQ ID NO: 63 Clone from FIG. 7 TTAAAATAGG GGAATGGCAG Clone 2-5 CACAAGAAGG SEQ ID NO: 64 Clone from FIG. 7 GCGAGCGCTG AGCATAGTGC Clone 2-6 CCAACGACAC SEQ ID NO: 65 Clone from FIG. 7 ATAGGGTACA CGCCGCCTCC Clone 2-7 GGTAGGGTGG SEQ ID NO: 66 Clone from FIG. 7 TTAAAATAGG GGAATGGCAG Clone 2-8 TAGGATGGGT SEQ ID NO: 67 Clone from FIG. 7 AGGGTGGTCC CAGGAATGGC Clone 2-9 TAGGATGGGT SEQ ID NO: 68 Clone from FIG. 7 AGGGTGGCCC CAGGAATGGC Clone 2-10 GGTAGGGTGG SEQ ID NO: 69 Clone from FIG. 7 TTAAAATAGG GGAATGGCAG Clone 2-11 GATGTGGCCC SEQ ID NO: 70 Clone from FIG. 7 AGAAGCATAA CACGACGTAC Clone 2-12 TAGGATGGGT SEQ ID NO: 71 Clone from FIG. 7 AGGGTGGTCC CAGGAATGGC Clone 2-13 GGAGATGCAG SEQ ID NO: 72 Clone from FIG. 7 GTACTGAGTA GGGAGTGTGC Clone 2-14 TAGGATGGGT SEQ ID NO: 73 Clone from FIG. 7 AGGGTGGTCC CAGGAATGGC Clone 1 AATCAAGGGC SEQ ID NO: 74 Clone from FIG. 8 TGGTGTTAAA GGTGATCGAC TAG Clone 2 AAGGGGAGCC SEQ ID NO: 75 Clone from FIG. 8 ATCACACAGG AGGTCGCTTC GCT Clone 3 AAAGGCATCA SEQ ID NO: 76 Clone from FIG. 8 CCTAGAGTTG CCGCCGATAC TTG Clone 4 GGGGATGTGC SEQ ID NO: 77 Clone from FIG. 8 GAAACTGGTG ACTATGCGGG TGC Clone 5 CGAAAGGAGC SEQ ID NO: 78 Clone from FIG. 8 CATCAACCTT GAAACGCCCG TCC Clone 6 CAGACGGGAG SEQ ID NO: 79 Clone from FIG. 8 CCATCGACAT AGAGGTGATT GCC Clone 7 AGGGAAAGCC SEQ ID NO: 80 Clone from FIG. 8 ATCACCTAGA CACATACAGC ATG Clone 8 ATAAGAAGCC SEQ ID NO: 81 Clone from FIG. 8 ATCATAGGGA CCTAGCTAGC CCC Clone 9 CCAACAGACG SEQ ID NO: 82 Clone from FIG. 8 GTAGCACAAC ACTAGTACTC TGG Clone 10 ACAGACGCCC SEQ ID NO: 83 Clone from FIG. 8 CTAGTAAACA ATAACCGATG GCC Clone 11 ATAGCTACTC SEQ ID NO: 84 Clone from FIG. 8 GCCAAGGGTG ACTTCTGCTA TTG Clone 12 ATGGGGCAAC SEQ ID NO: 85 Clone from FIG. 8 GCGGAGACCT GTCGGTACTG CCT Clone 13 GCAATATAGC SEQ ID NO: 86 Clone from FIG. 8 ACTAAGCCTT AACTCCATGG TGG Clone 14 GCAAGGAAAA SEQ ID NO: 87 Clone from FIG. 8 ACAAGCAAGC CATCACGACC TAG Clone 15 CAGGCATCCC SEQ ID NO: 88 Clone from FIG. 8 AAGAAGTGTC AGCCGTTTCG TGG Clone 16 CAACAGGAGA SEQ ID NO: 89 Clone from FIG. 8 GCCCGACACA CAGATCTGGC CCC Clone 17 ACAAGCCATC SEQ ID NO: 90 Clone from FIG. 8 ACGTGAATGC CGACCGGTAC TGT Clone 18 ACCGACAAAC SEQ ID NO: 91 Clone from FIG. 8 AAGTCAATAC GGGACACGAT CCT Clone 19 CAGTGGGTCG SEQ ID NO: 92 Clone from FIG. 8 GGTCACAGCC ATGAGTGTTG CTG Clone 20 AACGGGAAAG SEQ ID NO: 93 Clone from FIG. 8 CCATCACCAT ATTTATCGTC CTG Clone 21 ACGGGCGCAA SEQ ID NO: 94 Clone from FIG. 8 ACAAGATGTA CAAAAGCATG GTG Clone 22 AGCGGGATAG SEQ ID NO: 95 Clone from FIG. 8 GGAACTATCG GACAATCGTC GTG Clone 23 GAGGATAAAA SEQ ID NO: 96 Clone from FIG. 8 GCCATCAACT AGAATGCGCA TGG 

What is claimed is:
 1. A method for simultaneously selecting at least two aptamers from a plurality of oligonucleotides of unknown binding specificity, wherein the at least two aptamers comprise a first aptamer sequence and a second aptamer sequence, wherein the first aptamer sequence binds to a first epitope on a target molecule and the second aptamer sequence binds to a second epitope on the target molecule; the method comprising: (a) synthesizing a plurality of first nucleic acid constructs and second nucleic acid constructs, the first nucleic acid construct comprising: A-B-C-D; the second nucleic acid construct comprising: E-F-G-H; wherein: A, C, E, and G are each different DNA sequences from about 10 to about 30 nucleotides in length, A and C together comprising a sequence to prime a polymerase chain reaction for amplifying a first aptamer sequence, and E and G together comprising a sequence to prime a polymerase chain reaction for amplifying a second aptamer sequence; B is a single-stranded randomly synthesized oligonucleotide of unknown sequence that is from about 20 to about 110 nucleotides in length; D and H are a pair of complementary nucleotide sequences from about 2 to about 20 nucleotides in length, wherein D and H have a free energy for association from about 5.5 kcal/mole to about 8.0 kcal/mole at a temperature from approximately 21° C. to about 40° C. and at a salt concentration of approximately 1 mM to about 100 mM; and F is a single-stranded randomly synthesized oligonucleotide of unknown sequence that is from about 20 to about 110 nucleotides in length that contains specific sequences binding to a first epitope of the target molecule; (b) contacting a plurality of first nucleic acid constructs and a plurality of second nucleic acid constructs with a plurality of target molecules to form a mixture of nucleic acid constructs and target molecules, wherein the mixture comprises a complex of a target molecule, a first bound nucleic acid construct, and a second bound nucleic acid construct, wherein the first bound nucleic acid construct binds to a first epitope of the target and the second bound nucleic acid construct binds to a second epitope of the target molecule, (c) isolating from the mixture the complex comprising the target molecule having the first nucleic acid construct bound to the first epitope and the second nucleic acid construct bound to the second epitope; and (d) determining the sequence of B from the first bound nucleic acid construct and determining the sequence of F from the second bound nucleic acid construct, wherein the sequence of B is the first aptamer sequence and the sequence of F is the second aptamer sequence.
 2. The method of claim 1, wherein the target molecule is selected from the group consisting of an analyte, a prion, a protein, a nucleic acid, a lipid, a carbohydrate, a biomolecule, a macromolecular complex, a fungus, a virus, and a microbial organism.
 3. The method of claim 1, wherein the target molecule is a protein.
 4. The method of claim 1, wherein the plurality of nucleic acid constructs are contacted with the target molecule by incubation in a salt buffer, having a pH range from about 6.5 to about 8.5 at a temperature ranging from about 21° C. to about 40° C.
 5. The method of claim 1, wherein the complex is isolated from the mixture using a process selected from the group consisting of nitrocellulose filters, magnetic beads, and sepharose beads.
 6. The method of claim 1, further comprising separating the first and second nucleic acid constructs from the complex to form a mixture comprising the target molecule, the first nucleic acid construct and the second nucleic acid construct.
 7. The method of claim 6, wherein the first nucleic acid construct and the second nucleic acid construct are separated from the target molecule by denaturing the target molecule or incubating the complex with an excess of competitor.
 8. The method of claim 7, wherein the first nucleic acid construct and the second nucleic acid construct are purified from the mixture by precipitating the nucleic acid.
 9. The method of claim 7, further comprising amplifying the first nucleic acid construct and the second nucleic acid construct by the polymerase chain reaction to form a polymerase chain reaction product.
 10. The method of claim 9, wherein the first nucleic acid construct and the second nucleic acid construct are purified from the polymerase chain reaction product by agarose gel separation and purification of the nucleic acid from the gel.
 11. The method of claim 9, further comprising repeating the selection, separation, amplification and purification steps of the method from about 4 to about 20 times, wherein at the end of each cycle the first nucleic acid construct and the second nucleic acid construct comprising double-stranded nucleotide sequence have the double-stranded nucleotide sequence separated to form single stranded nucleotide sequence prior to the beginning of each cycle.
 12. The method of claim 11, wherein the double stranded nucleotide sequence is separated to single stranded nucleotide sequence by binding the nucleic acid to strepavidin linked beads and denaturing the nucleic acid.
 13. The method of claim 1, wherein A, C, E, and G are from about 15 to about 25 nucleotides in length.
 14. The method of claim 1, wherein A, C, E, and G are from about 15 to about 20 nucleotides in length.
 15. The method of claim 1, wherein A, C, E, and G are from about 16 to about 18 nucleotides in length.
 16. The method of claim 1, wherein A, C, E, and G are 18 nucleotides in length.
 17. The method of claim 1, wherein A, C, E, and G have an average GC content from about 55% to about 60%.
 18. The method of claim 1, wherein A, C, E, and G have an average GC content of about 60%.
 19. The method of claim 1, wherein B and F comprise nucleotide bases selected from the group consisting of DNA bases, RNA bases, modified DNA bases, and modified RNA bases.
 20. The method of claim 1, wherein B and F are from about 25 to about 75 nucleotides in length.
 21. The method of claim 1, wherein B and F are from about 30 to about 60 nucleotides in length.
 22. The method of claim 1, wherein D and H are from about 4 to about 15 nucleotides in length.
 23. The method of claim 1, wherein D and H are from about 5 to about 7 nucleotides in length.
 24. The method of claim 1, wherein D and H have a free energy of association of about 6.0 kcal/mole to about 8.0 kcal/mole.
 25. The method of claim 1, wherein D and H have a free energy of association of about 7.5 kcal/mole.
 26. The method of claim 1, wherein A, C, E and G are approximately 18 nucleotides in length; B and F are from about 30 to about 60 nucleotides in length; and D and H are from about 5 to about 7 nucleotides in length.
 27. The method of claim 26, wherein D and H have a free energy of association of about 7.5 kcal/mole.
 28. The method of claim 27, wherein A, C, E, and G have an average GC content of about 60%. 